Robert Klipper

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labelling preformed liposomes with technetium-99m (99mTc) has been developed which is simple to perform and stable in vivo. Previous 99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (greater than 90%) with excellent stability. A commercially available radiopharmaceutical kit--hexamethylpropyleneamine oxime (HM-PAO)--is reconstituted with 99mTcO4- and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity. This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with 99mTc may also have applications in diagnostic imaging.

Circulation Kinetics and Organ Distribution of Hb-Vesicles Developed as a Red Blood Cell Substitute

Phospholipid vesicles encapsulating concentrated human hemoglobin (Hb-vesicles, HbV), also known as liposomes, have a membrane structure similar to that of red blood cells (RBCs). These vesicles circulate in the bloodstream as an oxygen carrier, and their circulatory half-life times (t1/2) and biodistribution are fundamental characteristics required for representation of their efficacy and safety as a RBC substitute. Herein, we report the pharmacokinetics of HbV and empty vesicles (EV) that do not contain Hb, in rats and rabbits to evaluate the potential of HbV as a RBC substitute. The samples were labeled with technetium-99m and then intravenously infused into animals at 14 ml/kg to measure the kinetics of HbV elimination from blood and distribution to the organs. The t1/2 values were 34.8 and 62.6 h for HbV and 29.3 and 57.3 h for EV in rats and rabbits, respectively. At 48 h after infusion, the liver, bone marrow, and spleen of both rats and rabbits had significant concentrations of HbV and EV, and the percentages of the infused dose in these three organs were closely correlated to the circulatory half-life times in elimination phase (t1/2β). Furthermore, the milligrams of HbV per gram of tissue correlated well between rats and rabbits, suggesting that the balance between organ weight and body weight is a fundamental factor determining the pharmacokinetics of HbV. This factor could be used to estimate the biodistribution and the circulation time of HbV in humans, which is estimated to be equal to that in rabbit.

Dual Radiolabeled Liposomes: Biodistribution Studies and Localization of Focal Sites of Infection in Rats

Liposomes encapsulating both glutathione and deferoxamine were labeled with 99mTc-HMPAO and 111In-oxine at the same time. These dual radiolabeled liposomes were intravenously injected in rats with S. aureus infection in thigh. The target-to-background ratio (T/BG) increased from 2.9 at 2 h to 4.4 at 8 h in 99mTc images. In 111In images, T/BG of 5.5 at 8 h increased to 10.5 by 48 h. The 24-h spleen uptake of 111In- and 99mTc-liposomes was 24.14%ID and 8.91%ID. In femur, 99mTc-liposomes remained at approximately 10.5%ID, but 111In-liposomes increased from approximately 11%ID at 4 h to approximately 25.5%ID at 24 h. The simultaneous presence of 99mTc and 111In in the liposomes resulted in good early (2-8 h) as well as delayed (24-48 h) images delineating the infection site.

Circulation persistence and biodistribution of lyophilized liposome-encapsulated hemoglobin: an oxygen-carrying resuscitative fluid.

Objective: To evaluate the circulation persistence and organ biodistribution of a freezedried, oxygen carrying resuscitative fluid: liposome‐encapsulated hemoglobin. Design: Randomized, animal studies. Setting: Accredited animal research facilities. Subjects: Normal female Balb/c mice and male New Zealand rabbits. Interventions: Two groups of normal female Balb/c mice were injected in the tail vein with either lyophilized liposome‐encapsulated hemoglobin (n = 9) that was reconstituted just before administration, or with unlyoph ilized liposomeencapsulated hemoglobin (n = 9) as a comparison. Two groups of male New Zealand rabbits were injected in the ear vein with either lyophilized 99mTc‐liposome‐encapsulated hemoglobin (n = 6) or unly oph ilized 99mTc‐liposome‐encapsulated hemoglobin as a comparison (n = 6). After injection, mice were anesthetized by brief inhalation of halothane followed by blood sampling through the retro‐orbital sinus. Rabbits were anesthetized 30 mins before liposome‐encapsulated hemoglobin administration with an intramuscular injection of a 5:1 mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Rabbits were then dynamically imaged for 90 mins, housed, and at 20 hrs, imaging again followed by autopsy and tissue sampling to validate imaged organ biodistributions. Measurements: Circulation persistence in the mouse was measured by removing a blood sample at various time points up to 24 hrs after injection. The blood sample was centrifuged in a hematocrit capillary tube and the disappearance of the sedimented liposome‐encapsulated hemoglobin fraction was measured. The change in the sedimented fraction of the liposomes with time was used to generate circulation persistence profiles in mice. The circulation persistence and organ biodistribution of 99mTc‐liposome‐encapsulated hemoglobin was measured by circling regions of interest on computer‐generated gamma camera images. These image intensities were then calculated as a function of total injected dose which was measured from a known volume and activity of 99mTc‐liposomeencapsulated hemoglobin. Actual tissue uptake was estimated from images by subtracting blood pool contribution which was measured by injecting 99mTc‐labeled rabbit red cells. Imaged organ biodistribution was validated at 20 hrs by measuring activity in weighed portions of tissue after autopsy. Main Results: The mean circulation half‐life of liposome‐encapsulated hemoglobin in mice injected at a dose of 1.0 g phospholipid/kg mouse and 1.95 g hemoglobin/kg was approximately 10.4 ± 0.5 (sd) hrs. The circulation half‐life of lyophilized liposome‐encapsulated hemoglobin was 10.7 ± 0.7 hrs. The circulation profiles demonstrate a rapid removal phase over the first 4 hrs after injection, followed by a secondary slow removal measured up to 24 hrs. The rapid removal phase of liposome‐encapsulated hemoglobin and lyophilized liposome‐encapsulated hemoglobin in the rabbit (injected at the same dose) indicated that lyophilized liposome‐encapsulated hemoglobin persists longer than the unlyophilized form in the first 4 hrs after injection. The organ biodistributions of unlyophilized 99mTc‐liposome‐encapsulated hemoglobin and lyophilized 99mTc‐liposome‐encapsulated hemoglobin in the rabbit demonstrate that the reticuloendothelial system is the primary site of removal, with significant uptake of lyophilized 99mTc‐liposome‐encapsulated hemoglobin by the liver (15.6 ± 1.0%), bone marrow (12.6 ± 1.6%), and spleen (9.7 ± 1.1%). The kidneys showed little accumulation of unlyophilized 99mTc‐liposome‐encapsulated hemoglobin or lyophilized 99mTc‐liposome‐encapsulated hemoglobin (1.6 ± 0.2% and 1.8 ± 0.1%, respectively), an important result for the efficacy and safety of this hemoglobin‐based blood substitute. Conclusion: The present results suggest that liposome‐encapsulated hemoglobin (and lyophilized liposome‐encapsulated hemoglobin) have pharmacokinetics that enable oxygen delivery during early treatment for hemorrhagic shock. The organ biodistribution demonstrates that the monocyte phagocytic system is principally involved with the slow removal of liposomeencapsulated hemoglobin (and lyophilized liposome‐encapsulated hemoglobin) over the course of 24 hrs. The lyophilized liposome‐encapsulated hemoglobin has similar pharmacokinetics to freshly prepared liposome‐encapsulated hemoglobin and could be an important storage strategy for the utilization of liposome‐encapsulated hemoglobin in areas where stored blood is unavailable. (Crit Care Med 1994; 22:142‐150)

Evaluation of [99mTc] liposomes as lymphoscintigraphic agents: comparison with [99mTc] sulfur colloid and [99mTc] human serum albumin

Abstract This study investigates the use of [ 99m Tc] liposomes for the detection of sentinel lymph nodes. A variety of [ 99m Tc] liposome formulations were compared with common lymphoscintigraphic agents including [ 99m Tc] regular-sulfur colloid (SC), [ 99m Tc] 0.22 μ-filtered-SC, [ 99m Tc] reduced heating time 0.22 μ-filtered-SC, and [ 99m Tc] human serum albumin (HSA) in rabbits. Images were acquired for the first 60 minutes and at 24 hours, followed by tissue biodistribution study. All agents except [ 99m Tc] regular SC demonstrated good migration from the injection site. Agents were retained in the popliteal node at 24 hours to varying degrees as follows: both [ 99m Tc] filtered SC preparations > [ 99m Tc] regular SC > [ 99m Tc] liposomes > [ 99m Tc] HSA. [ 99m Tc] liposome imaging can be used to develop novel liposome compositions with improved lymph node diagnostic and drug delivery characteristics.

Repeat Injection Studies of Technetium-99M-Labeled Peg-Liposomes in the Same Animal

AbstractThis study was designed to determine if repeat injections of polyethylene glycol (PEG) coated liposomes in the same animal lead to a change in biodistribution due to a biological response. To evaluate the long-term storage of PEG-liposomes for imaging applications and to remove any concerns of batch variability, one large batch of PEG-liposomes containing glutathione and sucrose was prepared and used for the entire study. On each day that an imaging study was performed, an aliquot of PEG-liposomes were labeled with techne-tium-99m (99mTc) using hexamethylpropyleneamine oxime. Rabbits (n = 4) were injected with 99mTc-PEG-liposomes (2 ml; 13 mg phospholipid/kg body wt; 2 mCi 99mTc-activity) at 8 days after manufacture and imaged under a gamma camera. Heart-to-lung ratios, heart-to-liver ratios and organ activities were determined by region of interest analysis. Blood samples were collected to generate blood clearance curves. Six weeks later the same rabbits were given a second dose of 99mTc-PEG-lipo...

Pharmacokinetics and biodistribution of [111In]-avidin and [99mTc]-biotin-liposomes injected in the pleural space for the targeting of mediastinal nodes

Pharmacokinetics and mediastinal node uptake of [111In]-avidin and [99mTc]-biotin-liposomes following either intrapleural (pleural) or intraperitoneal (ip) injection were determined using scintigraphic imaging. Biodistribution results of [111In]-avidin at 44 h showed 3.3% uptake in mediastinal nodes by pleural injection vs 1.3% with ip injection. Mediastinal node accumulation with [99mTc]-biotin-liposomes was not different between injections (0.6% ip vs 0.5% pleural). This study demonstrates the potential of the pleural route as a technique for mediastinal node targeting using the avidin/biotin-liposome system.

Accumulation of PEG-liposomes in the Inflamed Colon of Rats: Potential for Therapeutic and Diagnostic Targeting of Inflammatory Bowel Diseases

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with severe toxicity related to the nonspecific and ubiquitous interaction of drugs with the organs and tissues. In order to prevent side effects from aggressive and prolonged treatment with glucocorticoids and immunosuppressive agents, preferential accumulation of these potent drugs in diseased tissue is desired. In this work, we report that liposomes show a remarkable tendency to accumulate in inflamed colon of rats with experimental colitis. The disposition of liposomes was monitored by labeling them with Tc-99m followed by gamma camera imaging, and determining biodistribution of radioactivity in various organs. The images showed distinct accumulation of radioactivity in the colon of rats with colitis, while the abdomen of normal rats was conspicuously free of any visible radioactivity. Although images acquired 4 h after Tc-99m-liposome injection were clear enough for diagnostic indication, the real potential of liposomes for drug delivery was evident in 24 h images where the major organs of liposome accumulation were dwarfed by intense colon activity in animals with colitis. On necropsy, 13.5% ± 5.48 of the activity accumulated in the inflamed colon as compared to only 0.1% in the normal colon, giving a target-to-nontarget ratio of 135. The blood borne radioactivity was 9% ± 2.12 (colitis) and 25.7% ± 4.27 (normal), indicating that the decrease in circulating liposomes is associated with an increase in liposome accumulation in the inflammatory site. The other two major organs that accumulated liposomes were spleen (10.7% normal vs. 11% colitis) and liver (8% normal vs. 10.1% colitis). In conclusion, this study demonstrates the innate propensity of liposomes to accumulate in the sites of inflammation and potential of liposomes loaded with therapeutic drugs or diagnostic agents for targeting colitis.

Physiological responses, organ distribution, and circulation kinetics in anesthetized rats after hypovolemic exchange transfusion with technetium-99m-labeled liposome-encapsulated hemoglobin.

ABSTRACT Physiological responses and circulation properties of liposome-encapsulated hemoglobin (LEH) labeled with technetium-99m (99mTc) were measured in rats after a 10% (170 mg/kg hemoglobin, 430 mg/kg phospholipid) or a 50% (450 mg/kg hemoglobin, 2.3 g/kg phospholipid) hypovolemic exchange transfusion (n = 5 per exchange group). Mean arterial pressure returned to baseline values (105 ± 8 mmHg) by 90 min post-infusion for both groups. By 20 h, mean arterial pressure remained at baseline values for the 10% group, but dropped to 30 ± 14 mmHg for the 50% group. For both groups, bradycardia was seen after the exchange period, but heart rate recovered by 30 min for the 10% group and by 90 min for the 50% group. The 99mTc-LEH remained in circulation longer for the 50% group (18.2 h half-life) than for the 10% group (2.4 h half-life). Removal of 99mTc-LEH from the bloodstream was via the liver and spleen. At 20 h, 99mTc-LEH accumulation in these organs was greater for the 10% group (liver, 36.2 ± 1.7%; spleen, 37.5 ± 2.5%) than for the 50% group (liver, 17.0 ± 1.4%; spleen, 17.1 ± 1.4%). The data show that there is less clearance of 99mTc-LEH from the bloodstream by the reticuloendothelial system after a 50% hypovolemic exchange transfusion, thus supporting the possible use of LEH as an oxygen-carrying resuscitative fluid in situations of severe blood loss.

[99mTc] Liposomes for localizing experimental colitis in a rabbit model

[(99m)Tc] liposomes (TL) and [(111)In]WBCs (WBCs) were compared for imaging colitis in rabbits. At 24 h, liver was the major organ of accumulation (7.8% TL vs 39.7% WBCs), besides blood (44.1% TL vs 24.4% WBCs) and spleen (0.3% TL vs 7% WBCs). The inflamed colon accumulated 37.1 times more of [(99m)Tc] liposomes than the normal colon whereas the ratio was only 15.2 in case of [(111)In] WBCs. The study demonstrates the usefulness of [(99m)Tc] liposomes for imaging inflammatory bowel diseases.